The studies outlined in this proposal are focused on the endocardium. Receptor tyrosine kinase (RTK) signaling is fundamental to the formation of blood vessels. Recent studies suggest that these molecules are also critically important during the reciprocal tissue interactions necessary for proper endocardial morphogenesis. This project focuses on the role of RTKs signaling: i) during the initial assembly of the endocardial tube, and ii) as a mediator of reciprocal signaling between the early endocardial and myocardial primordia. Both of these events coincide with embryonic stages when most cardiac developmental malformations are known to occur. Aim 1: To elucidate the role of VEGF/VEGF receptor signaling in endocardiogenesis. We will- a) Conduct in vivo experiments to determine the morphological consequences of either over-stimulating or neutralizing VEGF signaling. b) Conduct in vivo experiments to determine if the protrusive activity associated with VEGF/VEGF receptor signaling involves integrin- mediated ECM adhesions. Aim 2: To elucidate the role of Tie2/Angiopoietin signaling in endocardiogenesis we will- a) Conduct in vivo experiments to determine the morphological consequence of over-stimulating and inhibiting Tie2 receptor signaling. b) Determine the normal expression pattern of the Tie 2 receptor using mice expressing GFP under the control of Tie2 promoter, and investigate abnormal endocardiogenesis in mouse embryos lacking the Tie2 gene. Aim 3: to elucidate the role of ephrin-B2 signaling in endocardiogenesis we will- a) Analyze the phenotype of mice in which the ephrin-B2 gene has been abrogated. b) Analyze the affects of ephrin-B2 constitutive expression in vivo. c) Conduct in vivo experiments to determine the morphological consequences of increasing or neutralizing ephrin-B2 signaling. d) Knockout ephrin-B2 mRNA in avian embryos using ribozyme-based technology.